Working groups

Barrier function of the skin

The skin provides an essential barrier for our body. It prevents the invasion of pathogens, allergens and other xenobiotics as well as the loss of water and solutes. Impairment of skin barrier function can be cause of several skin diseases or aggravate them.

WG Brandner: The role of tight junctions in skin barrier function and the interaction of the various components of the skin barrier

The skin barrier is composed of several components: the mechanical barriers of the stratum corneum and the tight junctions (TJs), the microbiome barrier, the immunological barrier which comprises immune cells and cytokines, and the chemical barrier which comprises antimicrobial peptides. These barriers interact with each other and an alteration of a distinct barrier influences the others.

The skin barrier plays an important role in skin diseases. For example, it is known that a skin barrier defect is causative for atopic dermatitis and results in a vicious circle: Impaired skin barrier leads to increased uptake of allergens and increased colonialization/invasion of bacteria, this results in an (exaggerated) reaction of the immune system and cutaneous inflammation which intensifies barrier impairment. Also in cutaneous wounds, the complete loss of skin barrier function is a tremendous problem because it facilitates invasion of pathogens and dehydration of the wound which promotes the development of chronic wounds.

The focus of our work is the Tight Junction barrier and its interaction with the other skin barriers. TJs are cell-cell junctions which form a barrier in the stratum granulosum. In addition to interfollicular epidermis, TJs are also found in hair follicles. The loss of the TJ protein Claudin-1 in mice results in death at the first day of birth due to tremendous transepidermal water loss. TJs are composed of a variety of transmembrane proteins (families of claudins, TJ-associated MARVEL-proteins (TAMPs) and junctional adhesion molecules (JAMs)) as well as plaque proteins (e.g. zonula occludens proteins (ZOs), cingulin, MUPP1). We could further show that TJ proteins – besides their role in barrier function – are also involved in differentiation, proliferation, apoptosis and cell-cell adhesion of keratinocytes. These functions are, at least partly, mediated by TJ proteins which are not part of TJ structures, i.e. they are TJ structure-independent.

TJs interact with the stratum corneum, the immunological barrier, the microbiome, and the chemical barrier.

In several skin diseasesTJs and TJ proteins are altered. These diseases can be classified into diseases with a primary TJ defect, which means the alteration of a TJ protein is causative for the disease, and into diseases with a secondary TJ defect, which means TJ proteins are changed during the course of the disease and contribute to pathogenesis. NISCH (neonatal ichthyosis sclerosing cholangitis)-syndrome which is caused by a loss of the TJ protein Claudin-1 belongs to the first category, psoriasis vulgaris and ichthyosis vulgaris can be categorized into the second one. For atopic dermatitis a genetic correlation with Claudin-1 SNPs was observed in a Northern American cohort, but not in a European cohort. However, TJ proteins are altered secondarily in lesional skin of patients in European cohorts.

While barrier impairment is negative in skin diseases, it can be desired in certain situations such as topical drug delivery. A better understanding of the TJ barrier and its interaction with the other barriers will help in future to optimize active pharmaceutical ingredients (APIs) and their formulations for better uptake and efficacy. To test the penetration and effect of these APIs and their formulations we developed several model systems: 3D reconstructed human epidermis (RHE) consisting of keratinocytes only, 3D reconstructed human skin (RHS) consisting of keratinocytes and fibroblasts, and ex-vivo skin models (Hamburg model of penetration). These three models can be used complementary. The RHE and RHS have the advantage of being of human origin and easy manipulation, e.g. by siRNA mediated protein knock-down or by using patient cells. Further, they can be investigated at different stages of maturation. However, their skin barrier function is impaired compared to in vivo human skin also when completely maturated, and a correction factor has to be incorporated. The Hamburg model of penetration is of porcine origin, and manipulation e.g. by siRNA treatment is only rarely possible, but its skin barrier function has been shown to be very similar to human skin and penetration can be investigated into different parts of the epidermis and dermis.

Porcine and human ex-vivo wound healing models

This model is a patented full thickness wound healing model which is based on porcine or human skin. The model can be used for up to 5-7 days to investigate basic wound healing mechanisms or for the testing of active ingredients, cremes, gels, ointments, formulations, wound dressings or physical wound treatments. It comprises aspects of wound healing phase 1 (inflammatory phase) as well as phase 2 (regeneration phase).

In recent years we further developed this model to an infected ex-vivo wound healing model (see also services ).

Porcine ex-vivo infection model

This is a full thickness skin model that is infected with Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, or Candida albicans respectively. We use this model to elucidate the interaction of bacteria/fungi with the various components of the skin barrier, especially tight junctions. Further, the model can be used for testings concerning the antimicrobial /antifungal efficacy of substances and dressings (see also lab services ) in a context which does not only take the interaction of bacteria and the substances/dressings into account, but which also regards the interaction of both with the skin. This research is performed in collaboration with Prof. Holger Rohde (Department of Medical Microbiology, Virology and Hygiene). Recently, we used this model in non-infected form to investigate the effect of snake venoms on the skin.

Reconstructed human epidermis and reconstructed human skin can be investigated immunohistologically, biochemically, molecular biologically and functionally. They can be manipulated e.g. by knock-down of certain proteins or by the utilization of patient cells.

These either porcine or human full thickness models allow the investigation of penetration of substances (e.g. in different formulations) into different layers of the skin. We have shown that these models are viable and metabolically active, and they exhibit normal differentiation, tight junction barrier function and proliferation. Further, we demonstrated that human and porcine skin show similar penetration rates.

Infected porcine oral mucosa model

In this model, which is based on porcine oral mucosa, we investigate the efficacy of various antifungal drugs, antiseptics and washing lotions concerning the reduction of bacterial/fungal load, and also concerning the viability, differentiation, proliferation and barrier function of the mucosa.

Team

Pia Houdek (medical technical assistant)Germar Schüring (assistant)

New active ingredients, formulations and physical therapy options have to be tested preclinically concerning their efficacy and safety in appropriate test systems. Also optimization of formulations has to be performed in test systems, preferably without animal experiments. Moreover, due to REACH (European regulation for Registration, Evaluation, Authorization, and Restriction of Chemicals), the number of safety and toxicology testings which can not be performed in animal experiments is largely expanding. We develop test systems for skin, eye and mucosa.

Some of these test systems are provided as services to companies and institutes.

WG Zorn-Kruppa: Eye

Hemicornea model: This is a threedimensional (3D) artificial cornea model which is bioengineered by using immortalized human cell lines. The model consists of corneal fibroblasts seeded into a collageneous stromagel, which is subsequently overlayed by a multilayered epithelium of corneal epithelial cells. Based on this hemicornea model several test methods have been established to evaluate permeability and toxicity of substances.

Molecular Andrology

The true identity of human spermatogonial stem cells (hSSCs) could not be uncovered to date. On the other hand, therapeutical application of this cell type may be useful.

By microarray analysis, we identified the Fibroblast growth factor receptor 3 (FGFR3) as a human spermatogonial specific marker. FGFR3 is expressed on the cell surface of A type spermatogonia (SPG). Immunofluorescent multiple staining followed by confocal microscopy demonstrated the appearance of the protein only within non-proliferating and non-differentiating SPG and the coexpression with the pluripotency marker UTF1. Additionally, FGFR3 protein is only expressed in small spermatogonial clusters, as revealed by whole mount preparations of seminiferous tubules. Hence, a potential stem cell state of the FGFR3+ human SPG is indicated.

In our current project, we want to optimize culture conditions for by magnetic beads isolated FGFR3+ SPG to propagate the potential hSSCs in their undifferentiated state via self renewal. Therefore, we want to utilize established protocols for hSSC proliferation and own approaches with growth factors in different combinations and concentrations. Cellular expansion of the potential hSSCs should serve as a prerequisite for xenotransplantation which is the only approach to reveal hSSC characteristics. Analysis of the epigenetic state and marker expression of pre- and post-cultivated cells should identify and therefore exclude any cellular changes during cell culture.

The new findings are anticipated to gain knowledge about the mechanism of human spermatogenesis and may have implications for a therapeutical approach with hSSCs.

Team

Beate Roth (medical technical assistant)

Dr. Andrea Salzbrunn (physician)

Dr. Domenica Varwig-Janßen (physician)

WG Spiess: Molecular causes of male idiopathic infertility

In our research group we are investigating the gene expression (the turning on and off of genes) during spermatogenesis (maturation of germ cells) in the testis and in spermatozoa (sperm cells) of the male.Many men have a so-called “idiopathic infertility”, which means that the causes of their impaired spermatogenesis, which leads to a decreased sperm number in their ejaculates (semen), are largely unknown. We aim to uncover the mechanisms that have a negative influence on the differentiation of germ cells in the testis. However, some men present with a completely normal ejaculate, and although the female partner is inconspicuous at the gynecological level, pregnancy is not achieved within a considerate time. We investigate, if these men have molecular disorders in their sperm cells that are not evident in a normal spermiogram (analysis of semen quality) and that may exert a negative effect on the sperm-oocyte interaction.

Team

Tumor biology

Skin tumors are of high interest because they are frequent (e.g. incidence of basal cell carcinoma 100/100,000/year) or highly aggressive (malignant melanoma, Merkel cell carcinoma). We investigate several different skin tumors in basic and clinical research. In addition, we are engaged in the context of our infertility clinic with testis cancer.

Basic research

WG Moll / Brandner: Malignant melanoma

Malignant melanoma is the skin tumor with highest risk to metastasize. It is responsible for more than 90% of all deaths due to skin tumors. In 2008, 17,800 new melanoma cases were diagnosed and 2500 patients died from melanoma in Germany. Fair-skinned persons (skin type I and II) have a higher incidence to develop malignant melanoma than persons with skin type III and IV.

Malignant melanoma has to be distinguished from benign skin tumors, e.g. dysplastic nevi. This is done clinically as well as (immuno)-histochemically, but can be challenging in ambiguous cases.

Therapy depends on tumor stage and mutation status of the tumor and can include tumor excision, radiotherapy, adjuvant interferon therapy, signal transduction inhibitor therapy (BRAF, c-KIT), immunotherapy (Ipilimumab) as well as mono- and poly-chemotherapies. Especially in stage IV satisfying long term therapies are still missing. Thus, development/validation of new therapies but also prevention of tumorigenesis and progression are important aims of our research.

We are interested in the role of cell-cell and cell-matrix adhesion molecules as well as additional marker molecules in development and progression of malignant melanoma. One of our strategies is to establish cell cultures from tumors, manipulate them and investigate them concerning tumor growth and the formation of metastasis in SCID mice (collaboration with Prof. Schumacher institute of Anatomy and Experimental Morphology ). Further, in vitro investigation of metastasis is performed in 2D and 3D tumor invasion models.

Of special interest is also the identification of new markers to distinguish melanoma from benign skin tumors, especially in ambiguous cases, and the investigation of alterations of cell-adhesion molecules in the tumor microenvironment and its contribution to tumor progression.

Squamous cell carcinoma of the skin is a malignant tumor often growing with local destructions. However, it has only a small risk to metastasize (5%). It evolves often from non-malignant precursors and carcinoma in situ (e.g. actinic keratosis, Morbus Bowen). In addition, it can develop from chronically inflamed skin, e.g. chronic wounds, chronic inflammatory skin diseases and chronic radiodermatitis.

Chronic UV exposure is the most important etiological factor for the development of squamous cell carcinoma. In addition, carcinogenic substances, ironizing radiation, HPV, immunosuppression and genetic predisposition can play a role.

We are primarily interested in the processes leading from UV damaged skin to carcinoma in situ and squamous cell carcinoma and focus on cell-cell and cell-matrix adhesion molecules.

Merkel cell carcinoma (MCC) is a very aggressive neuroendocrine skin carcinoma. Its tumor specific 5 year survival rate is 60%. MCC is rare, but its incidence increased threefold during the last years, being now 0.3/100,000/year in fair-skinned European and American populations. MCC is mainly a tumor of the elderly (mean age ca. 70 years) and immunosuppressed persons. Localization is frequently at the head and neck area and the extremities, rarely at the trunk. Clinically, bluish-red tumors which grow rapidly can be seen. MCC tend to relapse in loco and metastasize in different organs in the course of the first two years. In approximately 80% of the tumors a newly described polyomavirus (Merkel cell polyomavirus) is responsible for tumorigenesis. Therapeutical options are excision, irraditation of the tumor and local lymph nodes as well has chemotherapies, which are, however, often only temporary successful. Thus, new therapeutic options are urgently needed.

We establish Merkel cell carcinoma cultures from tumor tissue in collaboration with the Dermatology Departments of the Universities of Erlangen and Frankfurt. The so-derived cell lines are characterized cell biologically and can be used for a variety of research approaches. In collaboration with Prof. Schumacher (Institute for Anatomy and Experimental Morphology) we investigate growth and formation of metastasis of these cells in SCID mice. In collaboration with Prof. Fischer (Department of Medical Microbiology, Virology and Hygiene) we perform investigations concerning the expression of the virus and its correlation to histological and clinical tumor parameters in vivo and in vitro. Finally, we investigate in collaboration with Prof. Pantel (Institute for tumor biology) single circulating tumor cells in peripheral blood.

Infertile men have an increased risk of developing germ cell cancer of the testis; this is specifically true for those showing primary impairment of spermatogenesis. At present approximately 20% of testis cancer patients are diagnosed during physical examination in preparation of a treatment for male infertility. Intratubular germ cell neoplasia (IGCNU) is the precursor lesion of testicular germ cell tumours which is also referred to as carcinoma in situ (CIS) or testicular intraepithelial neo­plasia (TIN). The precursor lesion is often present many years before the manifestation of an open tumour emphasizing its great significance for early detection of testis cancer.

Presently, testicular germ cell cancer and its precursor lesion IGCNU are diagnosed in testicular biopsies by histological and/or immmunohistochemical methods. Reliable markers for cancer cell detection are known and validated for long; the Oct3/4 transcription factor is specifically well suited as a robust nuclear marker of germ cell tumours. Our project aims at the development of improved diagnostic methods for patients attending consultation at an infertility clinic to safely and fast exclude IGCNU. Moreover, we are studying a novel nuclear structure specifically occurring in IGCNU to learn more about the development and progression of testis cancer. The focus of this study is on the nuclear envelope. A highly regulated barrier which mediates contact between the nucleus and the cytoplasm in eukaryotic cells, the nuclear envelope has been implicated in dynamic chromosome positioning and cytoskeletal interaction as well as chromatin organization and gene regulation. Its structure and composition in tumour cells differs from that of the corresponding normal cell type.

Team

Wound healing

In our working groups we are interested in basic mechanisms of wound healing but also in the development and validation of new therapies, e.g. with transplanted keratinocytes or natural plant products.

The first phase results in coagulation, cleaning of the wound, formation of a provisional matrix and promotion of wound regeneration. This is achieved by the activation of platelets and coagulation factors, migration of macrophages, neutrophils and other immune cells into the wound site and release of reactive oxygen species, proinflammatory cytokines and growth factors.

In the second phase, epidermis and dermis regenerate by proliferation and migration of keratinocytes and fibroblasts, the formation of new blood vessels and the generation of extracellular matrix. During this phase the wound is repelnished with granulation tissue and closed by a new epithelium.

The third phase is important for the further restoration of functionality of the skin (barrier function, tensile strength, sensitivity). It is characterized by a remodeling of extracellular matrix, reduction of myofibroblasts and blood vessels by apoptosis as well as atrophy of the hypertrophic epidermis. Tyically this remodeling results in a more or less pronounced scar.

Tight Junctions (TJs) are barrier-forming cell-cell junctions. In addition, TJ proteins are involved in cell proliferation, differentiation, cell-cell adhesion and apoptosis. During wound healing, TJ proteins are re-expressed very early in the regenerating epidermis. We investigate the role of TJ proteins in acute and chronic wounds in normal as well as diabetic skin.

Gap junctions are channel forming cell-cell junctions which mediate direct cell-cell communication. They allow the controlled exchange of ions, second messenger molecules and metabolites with a molecular weight of up to 1000 Da. Gap junctions are formed by connexins, a family of transmembrane proteins. We and others could show that gap junctions / connexins play an important role in wound healing. They are involved in migration, proliferation, differentiation and apoptosis as well as cytokine release. Of note, diabetic origin shows an influence on connexins and their susceptibility to external manipulation. In our present work we investigate the interplay of connexins with other cell junctions and signals influencing gap junctions during wound healing.

Team

Applied Science

WG Brandner: Investigation of natural products in wound healing

We are interested in the elucidation of the wound healing potential of natural products and the underlying mechanisms. For example, we could prove in a collaboration project with the Universitiy of Tübingen, the University of Freiburg and the Birken AG that an extract from birch bark has a positive effect on wound healing and reveal the underlying mechanisms. These investigations accompanied the process of drug development from the first preclinical investigations to clinical studies.

In another project, the positive effect of plantago major, a plant used in traditional medicine of several countries could be shown.

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